In all mammalian species studied so far, mature Leydig cells originate from primitive mesenchymal precursors. This process of differentiation involves a change in the cytologic phenotype from a fibroblastic-type mesenchymal cell to an epithelioid polyhedral Leydig cell with extensive development of smooth endoplasmic reticulum and steroid-type mitochondria, as well as a full complement of steroidogenic enzymes and membrane receptors for LH-hCG. However, there is little information in the literature regarding the characteristics of this process and the mechanisms involved in regulating Leydig cell differentiation. in this proposal, we intend to isolate, characterize, and culture Leydig cell mesenchymal precursors which will be obtained from the testes of rats previously treated with ethylene dimethanesulphonate (EDS), which destroys mature Leydig cells without affecting the capacity of the remaining stem cells to undergo differentiation. The isolation procedure will be performed at various time intervals after EDS administration. In this way, mesenchymal precursors of varying degrees of differentiation will be obtained. The cells will be cultured on reconstituted basement membrane (Matrigel) which is known to regulate important biological functions. In this model, the potential role of hormones and trophic factors in the differentiation process will bc analyzed. A coculture system using bicameral chambers with Sertoli cells grown in the upper chamber and mesenchymal precursors in the bottom one will bc utilized to explore the paracrine interactions between both cell types. Special attention will be given to morphological characteristics (light and electron microscopy), the presence of membrane LH receptors (immunocytochemistry and binding studies), cAMP responsiveness to LH, the presence of steroidogenic enzymes (immunocytochemistry of 3beta-HSD), and production of androgens. The experiments outlined in this proposal should provide new data on the factors involved in the process of Leydig cell proliferation and differentiation and on the basic biology of the Leydig cell - Sertoli cell interdependence in the testis.